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Monday, December 19, 2016

Gibson Assembly® PBnJ™ Overhang Extension and Sequence Insertion

Using the Gibson Assembly® method to introduce overhangs
and insertions

In the Gibson Assembly® Cloning Guideand our last blog
post, we introduced a variation of the
Gibson Assembly® method that does not rely on the use
of homologous overlapping ends for fragment assembly. This technique, Gibson
Assembly® PBnJ™ Cloning, has many potential
applications. Here, we’d like to focus on two of these, adding DNA overhangs
and insertions.

Creating overhangs

To create a 3’ overhang, simply design a primer
containing the intended overhang sequence. To protect the primer from chew-back
during the assembly process, include four phosphorothioate
modifications at the 3’ end of the primer. Creation of a fragment with the 3’
overhang is initiated by combining the single phosphorothioate-modified primer,
your corresponding DNA fragment-of-interest, and Gibson Assembly® Ultra Master Mix A. The Gibson Assembly® Ultra procedure yields a DNA fragment
containing a 3’ overhang, as shown in the following illustration.

Inserting DNA sequence between fragments during assembly

Insertions
can be added during cloning using another variation of this technique. Mutagenesis,
promoter or enhancer functional analysis, and large-scale genome modification studies,
or even simply adding a short sequence of interest (i.e. gRNA target sequence, barcode,
restriction sites, etc.) are all potential applications of Gibson Assembly® PBnJ™ Sequence Insertion Cloning. Gibson
Assembly® PBnJ™ Sequence
Insertion Cloning adds sequence between adjoining fragments during assembly. As
shown below, appropriately designed primers are critical to the outcome of the
assembly reaction. One primer is designed to contain homology to one of the
fragments, and another primer is designed with homology to the other fragment.
The insertion sequence must be added to the 3’ end of both primers and is
necessary to bridge the fragments to be joined since this is the only region
containing homology between the fragments. Both primers are synthesized with at
least four phosphorothioate bonds at the 3’ termini to protect them from
chew-back during the assembly reaction. The assembly reaction is initiated by
combining DNA fragments, appropriate phosphorothioate-modified primers, and Gibson
Assembly® Ultra Master Mix
A. Following 3’ chew-back mediated by Master Mix A, the reaction undergoes heat
inactivation and denaturation, which allows for the annealing of the
bridge/insertion region of the primers. Strand extension is then mediated by
Ultra Master Mix B resulting in seamless assembly of fragments with an
insertion.